Lespedeza bicolor Turcz., which belongs to the family Fabaceae, is a drought-tolerant plant that is helpful for wind prevention, sand fixation, water and soil conservation, and improving ecosystem. However, the plant currently lacks genomic or transcriptomic resources, and few effective expressed sequence tag-derived simple sequence repeat (EST-SSR) markers have been developed. To our knowledge, this is the first study to reveal the transcriptome sequence of this species and annotate the transcriptome and developed a set of EST-SSR markers for evolutionary studies. Approximately 64.76 million clean reads from the leaves of L. bicolor were generated using transcriptome sequencing by using Illumina sequencing technology, and 120 913 unigenes with mean length of 608 bp and N50 of 978 bp were obtained. The function of a total of 72 613 (60.05%) unigenes was annotated, and 25 658 and 28 950 unigenes could be aligned to the KEGG and KOG database, respectively. Search against the Gene Ontology (GO) led to the assignment of 47 561 unigenes to 55 different functional pathway categories, including three major pathways, i.e., biological process, cellular component, and molecular function. In addition, a total of 13 551 potential EST-SSR sites were identified. The EST-SSR sites identified might provide additional resources for developing molecular markers for further evolutionary studies on this species and congeners. The 173 selected EST-SSRs were used to amplify 56 markers with the expected size in L. bicolor and nine other Lespedeza species. The transcriptome dataset reported in this study might serve as an important public information platform for comparative and functional genomic as well as gene expression studies in L. bicolor and other closely related species. These EST-SSR markers provide a powerful tool for assessing the genetic diversity and population structure, constructing a DNA fingerprint database, generating genetic maps, predicting molecular marker-assisted breeding, and preserving genetic information.